The differentiation of Phytophthora species that are pathogenic on potatoes by an asymmetric PCR combined with single-strand conformation polymorphism analysis

The goal of this study was to develop a polymerase chain reaction (PCR) capable of differentiating Phytophthora species that are pathogenic on potatoes using a single primer pair. To achieve this objective, primers were derived from conserved regions flanking variable sequences in the internal transcribed spacer 1 (ITS1) of Phytophthora species. One primer pair produced a 140 bp product from P. infestans , P. erythroseptica and P. nicotianae . The PCR products were purified and used in an asymmetric PCR (A-PCR) protocol to generate single-strand DNA (ssDNA). The ssDNA of the Phytophthora potato pathogens reproducibly migrated in non-denaturing polyacrylamide gels in a species-specific manner.     

Effect of methyljasmononate on induction of late-blight resistance of potatoes using arachidonic acid

Methyl ester of jasmonic acid (Me-JA) influences the induced resistance of potato tubers to late blight caused by Phytophthora infestans. Treatment of potato tuber disk surface with Me-JA solution or exposure to an atmosphere containing Me-JA vapors (10(-6)-10(-5) M) increased the rate of rishitin biosynthesis induced by arachidonic acid or P. infestans. Methyl jasmonate increased the sensitivity of potato tissue to arachidonic acid. As a result, in the presence of Me-JA, the protective properties of arachidonic acid were observed at lower concentrations than in the absence of Me-JA. In addition, Me-JA reduced the adverse effects of lipoxygenase inhibitors (salicylhydroxamic acid and esculetin) on the induced resistance of potato tubers to late blight. Therefore, the synergistic interaction of Me-JA and biogenic elicitors can be regarded as part of a mechanism of potato defense against diseases.     

Rapid Detection of Phytophthora nicotianae in Infected Tobacco Tissues and Soil Samples Based on Its Ypt1 Gene

This paper describes the development of a polymerase chain reaction (PCR) assay for the detection of Phytophthora nicotianae , the causal agent of Phytophthora blight of tobacco and other plants. The PCR primers were designed based on a Ras-related protein ( Ypt 1) gene, and 115 isolates representing 26 species of Phytophthora and 29 fungal species of plant pathogens were used to test the specificity of the primers. PCR amplification with species-specific (Pn) primers resulted in a product of 389 bp only from isolates of P. nicotianae . The detection sensitivity with Pn primers was 1 ng of genomic DNA. Using Ypt 1F/ Ypt 1R as first-round amplification primers, followed by a second round using the primer pair Pn1/Pn2, a nested PCR procedure was developed, which increased the detection sensitivity 100-fold to 10 pg. PCR with the Pn primers could also be used to detect P. nicotianae from naturally infected tobacco tissues and soil. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring as well as guide plant disease management.     

Transgenic Potatoes Expressing a Novel Cationic Peptide are Resistant to Late Blight and Pink Rot

Potato is the world's largest non-cereal crop. Potato late blight is a pandemic, foliar wasting potato disease caused by Phytophthora infestans, which has become highly virulent, fungicide resistant, and widely disseminated. Similarly, fungicide resistant isolates of Phytophthora erythroseptica, which causes pink rot, have also become an economic scourge of potato tubers. Thus, an alternate, cost effective strategy for disease control has become an international imperative. Here we describe a strategy for engineering potato plants exhibiting strong protection against these exceptionally virulent pathogens without deleterious effects on plant yield or vigor. The small, naturally occurring antimicrobial cationic peptide, temporin A, was N-terminally modified (MsrA3) and expressed in potato plants. MsrA3 conveyed strong resistance to late blight and pink rot phytopathogens in addition to the bacterial pathogen Erwinia carotovora. Transgenic tubers remained disease-free during storage for more than 2 years. These results provide a timely, sustainable, effective, and environmentally friendly means of control of potato diseases while simultaneously preventing storage losses.     

A genetic analysis of quantitative resistance to late blight in potato: towards marker-assisted selection

Late blight caused by the oomycete Phytophthora infestans is the most important fungal disease in potato cultivation worldwide. Resistance to late blight is controlled by a few major genes (R genes) which can be easily overcome by new races of P. infestans and/or by an unknown number of genes expressing a quantitative type of resistance which may be more durable. Quantitative resistance of foliage to late blight was evaluated in five F₁ hybrid families originating from crosses among seven different diploid potato clones. Tuber resistance was evaluated in four of the families. Two of the families were scored for both foliage maturity and vigour. The five families were genotyped with DNA-based markers and tested for linkage with the traits analysed. QTL (quantitative trait locus) analysis identified at least twelve segments on ten chromosomes of potato having genes that affect reproducibly foliage resistance. Two of those segments also have major R genes for resistance to late blight. The segments are tagged by 21 markers that can be analyzed based on PCR (polymerase chain reaction) with specific oligonucleotide primers. One QTL was detected for tuber resistance and one for foliage vigour. Two QTLs were mapped for foliage maturity. Major QTL effects on foliage and tuber resistance to late blight and on foliage maturity and vigour were all linked with marker GP179 on linkage group V of potato. Plants having alleles at this QTL, which increased foliage resistance, exhibited decreased tuber resistance, later maturity and more vigour.     

Factors affecting the field infection of potato tubers of different cultivars by blight (Phytophthora infestans)

The relationships between rain and blight (Phytophthora infestans) were studied in unsprayed crops of cultivars differing widely in foliage and tuber susceptibility. The occasions when tubers were infected depended on rain and not cultivar, but numbers of tubers infected after rain was affected by the blight susceptibility of the cultivar. Infected tubers were first found when less than 5 % (BMS key) of the potato foliage was infected but few fresh infections occurred when 50–75% of the foliage had been destroyed. Some tubers were infected after 8 mm rain (tubers near the surface with even less) but large increases in numbers of tubers infected usually occurred only after 25 mm or more had increased soil moisture to above ‘field capacity’ around the tuber for at least 24 h. The most susceptible cultivars Ulster Ensign and Arran Banner had all plants with some tuber blight, and some plants with all tubers affected and often many lesions per tuber. Cultivars of intermediate susceptibility, King Edward and Up-to-Date, had some plants without blighted tubers, many with a few and very few with all. The more resistant cultivars Majestic and Arran Viking had many plants without infected tubers and many lesions that aborted while still necrotic threads, so that the fungus did not spread. Most infections occurred through tuber eyes, lenticels or sometimes growth cracks. The distribution of blight lesions on tubers differed in the different seasons, for example, lenticels were most commonly infected on Arran Banner and Ulster Ensign and eyes on King Edward, Majestic and Arran Viking. In late or slowly developing attacks, lesions on stems became more numerous and larger than in fast, early attacks and were prolific sources of spores on King Edward and Up-to-Date but not on Majestic and Arran Viking. Because much rain water runs down the stems of Up-to-Date and King Edward, stem lesions can provide an important source of inoculum for tubers.     

马铃薯非共生血红蛋白基因StHb1的克隆及表达

采用RT-PCR技术成功分离了马铃薯StHb1基因序列.经半定量RT-PCR分析表明,StHb1基因的表达在抗性品种(陇薯三号)和感性品种(荷兰十五)块茎中均受致病疫霉的侵染所抑制;StHb1基因在正常生长的马铃薯块茎组织中表达量最高;外源NO和H2O2的作用可明显地抑制StHb1基因的表达,但在抗性品种中该基因受抑制的程度低于感性品种.上述试验结果暗示了StHb1基因与马铃薯对致病疫霉侵染的抗性应答具有一定的相关性.     

The plant pathogen Phytophthora andina emerged via hybridization of an unknown Phytophthora species and the Irish potato famine pathogen, P. infestans

Emerging plant pathogens have largely been a consequence of the movement of pathogens to new geographic regions. Another documented mechanism for the emergence of plant pathogens is hybridization between individuals of different species or subspecies, which may allow rapid evolution and adaptation to new hosts or environments. Hybrid plant pathogens have traditionally been difficult to detect or confirm, but the increasing ease of cloning and sequencing PCR products now makes the identification of species that consistently have genes or alleles with phylogenetically divergent origins relatively straightforward. We investigated the genetic origin of Phytophthora andina, an increasingly common pathogen of Andean crops Solanum betaceum, S. muricatum, S. quitoense, and several wild Solanum spp. It has been hypothesized that P. andina is a hybrid between the potato late blight pathogen P. infestans and another Phytophthora species. We tested this hypothesis by cloning four nuclear loci to obtain haplotypes and using these loci to infer the phylogenetic relationships of P. andina to P. infestans and other related species. Sequencing of cloned PCR products in every case revealed two distinct haplotypes for each locus in P. andina, such that each isolate had one allele derived from a P. infestans parent and a second divergent allele derived from an unknown species that is closely related but distinct from P. infestans, P. mirabilis, and P. ipomoeae. To the best of our knowledge, the unknown parent has not yet been collected. We also observed sequence polymorphism among P. andina isolates at three of the four loci, many of which segregate between previously described P. andina clonal lineages. These results provide strong support that P. andina emerged via hybridization between P. infestans and another unknown Phytophthora species also belonging to Phytophthora clade 1c.     

THE REACTION OF VIRUS-INFECTED POTATO PLANTS TO PHYTOPHTHORA INFESTANS

The growth of Phytophthora infestans was retarded on leaves of potato plants that had been artificially inoculated with virus X or with virus Y.
Using different virus strains and potato varieties, the effect of virus infection on blight development was found to be greater, the more severe the systemic virus symptoms exhibited on the infected leaves before P. infestans inoculation.
The development of the fungus was never increased by virus infection.
The reduced blight development on virus-infected leaves is partially caused by an increase of resistance to infection. It is also suggested that virus infection alters the nutritional status of leaves to one less favourable for the development of P. infestans.     

BACTERIAL WET ROT OF POTATO TUBERS FOLLOWING PHYTOPHTHORA INFESTANS

Potato tubers infected with Phytophthora infestans in the field produce abnormal amounts of liquid which often appear on the surface of tubers kept in a saturated atmosphere. Under these conditions a soft rot, associated with bacteria, develops.
Healthy tubers artificially infected with pure cultures of P. infestans produce a similar liquid and, if further inoculated with pure cultures of certain bacteria, develop a wet rot which spreads if the bacteria are pathogenic or is confined to the zone of fungal invasion if the bacteria are saprophytes.
Sap extracted from Phytophthora -infected tissue contains more sugar and has a greater osmotic pressure than sap extracted from healthy tissue. Thus, water may be withdrawn from healthy tissue which would result in the infected tissue containing more liquid than does the healthy tissue.     

cDNA文库与RACE方法结合克隆一个马铃薯病程相关蛋白基因cDNA

为克隆马铃薯晚疫病抗性相关基因,深入研究马铃薯晚疫病抗性机制,应用SMART LD—PCR技术,以晚疫病菌混合小种诱导48h的水平抗性马铃薯(Solanum tuberosum L．)(R—gene—free)叶片为材料,构建了一个富集晚疫病抗性相关基因的cDNA文库。为提高克隆全长cDNA的效率,将cDNA文库与RACE技术结合,依据本实验室得到的病原诱导表达片段测序结果,在其内部设计两个特异引物,与文库载体臂上的通用引物配对,以文库噬菌体DNA为模板,用高保真PCR分别扩增出cDNA5′端与3′端,从而简便、快捷地得到全长cDNA序列。采用此方法,在马铃薯中克隆了一个受晚疫病菌诱导表达的cDNA,该cDNA长904bp,5′端有29bp的非翻译区,3′端具有完整的polyA尾,包含一个678bp的完整开放阅读框架,编码226个氨基酸(GenBank登录号：AY 185207)。BLAST检索发现其氨基酸序列与烟草一个新的病程相关蛋白基因NtPRp27具有90％的同源性,在马铃薯中尚未发现与之同源的已知基因。Northern杂交结果表明,水杨酸(SA)、茉莉酸(JA)、茉莉酸甲酯(MeJA)、机械伤害和渗透胁迫都能诱导该基因表达。该基因可能是马铃薯一个新的病程相关蛋白基因。     

Effect of methyl jasmonate on arachidonic acid-induced resistance of potato to late blight

Methyl ester of jasmonic acid (Me-JA) influences the induced resistance of potato tubers to late blight caused byPhytophthora infestans. Treatment of potato tuber disk surfaces with Me-JA solution or exposure to an atmosphere containing Me-JA vapors (10⁻⁶–10⁻⁵ M) increased the rate of rishitin biosynthesis induced by arachidonic acid orP. infestans. Methyl jasmonate increased the sensitivity of potato tissue to arachidonic acid. As a result, in the presence of Me-JA, the protective properties of arachidonic acid were observed at lower concentrations than in the absence of Me-JA. In addition, Me-JA reduced the adverse effects of lipoxygenase inhibitors (salicylhydroxamic acid and esculetin) on the induced resistance of potato tubers to late blight. Therefore, the synergistic interaction of Me-JA and biogenic elicitors can be regarded as part of a mechanism of potato defense against diseases.     

Families of short interspersed elements in the genome of the oomycete plant pathogen, Phytophthora infestans

The first known families of tRNA-related short interspersed elements (SINEs) in the oomycetes were identified by exploiting the genomic DNA sequence resources for the potato late blight pathogen, Phytophthora infestans. Fifteen families of tRNA-related SINEs, as well as predicted tRNAs, and other possible RNA polymerase III-transcribed sequences were identified. The size of individual elements ranges from 101 to 392 bp, representing sequences present from low (1) to highly abundant (over 2000) copy number in the P. infestans genome, based on quantitative PCR analysis. Putative short direct repeat sequences (6-14 bp) flanking the elements were also identified for eight of the SINEs. Predicted SINEs were named in a series prefixed infSINE (for infestans-SINE). Two SINEs were apparently present as multimers of tRNA-related units; four copies of a related unit for infSINEr, and two unrelated units for infSINEz. Two SINEs, infSINEh and infSINEi, were typically located within 400 bp of each other. These were also the only two elements identified as being actively transcribed in the mycelial stage of P. infestans by RT-PCR. It is possible that infSINEh and infSINEi represent active retrotransposons in P. infestans. Based on the quantitative PCR estimates of copy number for all of the elements identified, tRNA-related SINEs were estimated to comprise 0.3% of the 250 Mb P. infestans genome. InfSINE-related sequences were found to occur in species throughout the genus Phytophthora. However, seven elements were shown to be exclusive to P. infestans.     

Tagging QTLs for late blight resistance and plant maturity from diploid wild relatives in a cultivated potato (Solanum tuberosum) background

Phytophthora infestans causes an economically important disease of potato called late blight. The epidemic is controlled chemically but resistant potatoes can become an environment-friendly and financially justified alternative solution. The use of diploid Solanum tuberosum derived from European tetraploid cultivars enabled the introgression of novel genes encoding foliage resistance and tuber resistance from other species into the modern cultivated potato gene pool. This study evaluated the resistance of the obtained hybrids, its quality, expression in leaflets and tubers and its relation to the length of vegetation period. We also identified genetic loci involved in late blight resistance and the length of vegetation period. A family of 156 individuals segregating for resistance to late blight was assessed by three laboratory methods: detached leaflet, tuber slice and whole tuber test, repeatedly over 5 years. Length of vegetation period was estimated by a field test over 2 years. The phenotypic distributions of all traits were close to normal. Using sequence-specific PCR markers of known chromosomal position on the potato genetic map, six quantitative trait loci (QTLs) for resistance and length of vegetation period were identified. The most significant and robust QTL were located on chromosomes III (explaining 17.3% of variance observed in whole tuber tests), IV (15.5% of variance observed in slice tests), X (15.6% of variance observed in leaflet tests) and V (19.9% of variance observed in length of vegetation period). Genetic characterization of these novel resistance sources can be valuable for potato breeders and the knowledge that the most prominent QTLs for resistance and vegetation period length do not overlap in this material is promising with respect to breeding early potatoes resistant to P. infestans. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Purification of an isoform of patatin with antimicrobial activity against Phytophthora infestans.

Phytophthora infestans (Mont.) de Bary is infamous as the causal agent of the late blight epidemic contributing to the Irish potato famine of the mid 19th century and remains agriculture's most destructive disease as new mutations and migrations confound control measures. In efforts to develop resistant varieties, a somatic hybrid (the Wisconsin J series) between potato (Solanum tuberosum) and a wild relative (Solanum bulbocastanum) has been found to convey durable resistance against the pathogen. We screened the total protein (100 microg ml(-1)) of somatic hybrid varieties J138, J138A12, J101K12, J103K12, and J101K9 for in vitro spore germination inhibition of P. infestans. Since J138 exhibited maximum inhibition at 150 microg ml(-1) in comparison to other varieties, we purified a 40 kD protein from J138 tubers by assaying its ability to inhibit spore germination in P. infestans spores. The highly purified protein was able to inhibit P. infestans spore germination by 70% at the 2.5 microg ml(-1) concentration. The N-terminal sequence of this protein was found to have exact amino acid homology to patatin, the major storage protein of potato tubers. The inhibitory protein has the same molecular weight as patatin and cross-reacts with patatin antibodies. The infection of J138 plants with spores of P. infestans under greenhouse conditions showed that patatin is expressed in stem tissue 72 h after the plant is inoculated with field isolates of P. infestans (US8). In this communication, we report the purification, characterization and antifungal activity against spores of P. infestans of patatin-J from potato tubers.     

Disease resistance conferred by expression of a gene encoding H2O2-generating glucose oxidase in transgenic potato plants.

Plant defense responses to pathogen infection involve the production of active oxygen species, including hydrogen peroxide (H2O2). We obtained transgenic potato plants expressing a fungal gene encoding glucose oxidase, which generates H2O2 when glucose is oxidized. H2O2 levels were elevated in both leaf and tuber tissues of these plants. Transgenic potato tubers exhibited strong resistance to a bacterial soft rot disease caused by Erwinia carotovora subsp carotovora, and disease resistance was sustained under both aerobic and anaerobic conditions of bacterial infection. This resistance to soft rot was apparently mediated by elevated levels of H2O2, because the resistance could be counteracted by exogenously added H2O2-degrading catalase. The transgenic plants with increased levels of H2O2 also exhibited enhanced resistance to potato late blight caused by Phytophthora infestans. The development of lesions resulting from infection by P. infestans was significantly delayed in leaves of these plants. Thus, the expression of an active oxygen species-generating enzyme in transgenic plants represents a novel approach for engineering broad-spectrum disease resistance in plants.     

Inhibitory effect of a defensin gene from the Andean crop maca (Lepidium meyenii) against Phytophthora infestans

In this study, we report the isolation of a defensin gene, lm-def, isolated from the Andean crop 'maca' (Lepidium meyenii) with activity against the pathogen Phytophthora infestans responsible of late blight disease of the potato and tomato crops. The lm-def gene has been isolated by polymerase chain reaction (PCR) using degenerate primers corresponding to conserved regions of 13 plant defensin genes of the Brassicaceae family assuming that defensin genes are highly conserved among cruciferous species. The lm-def gene belongs to a small multigene family of at least 10 members possibly including pseudogenes as assessed by genomic hybridization and nucleotide sequence analyses. The deduced mature Lm-Def peptide is 51 amino acids in length and has 74-94% sequence identity with other plant defensins of the Brassicaceae family. The Lm-Def peptide was produced as a fusion protein using the pET-44a expression vector and purified using an immobilized metal ion affinity chromatography. The recombinant protein (NusA:Lm-Def) exhibited in vitro activity against P. infestans. The NusA:Lm-Def protein caused growth inhibition and hyphal damage at concentration not greater than 0.4 microM. In contrast, the NusA protein alone expressed and purified similarly did not show any activity against P. infestans. Therefore, these results indicate that the lm-def gene isolated from maca belong to the plant defensin family with activity against P. infestans. Its expression in potato, as a transgene, might help to control the late blight disease caused by P. infestans with the advantage of being of plant origin.     

The effect of Pythium oligandrum and chitosan used in control of potato against late blight and the occurrence of fungal diseases on tuber peel

The aim of two year investigation was the valuation the effect of biopreparate Polyversum (B.A.S. Pythium oligandrum) and preparate Biochikol 020 PC (B.A.S. chitosan) used in control of potato against Phytophthora blight on the tuber infestation during storage by Helminthosporium solani and tuber infestation by sclerotia of Rhizoctonia solani. As the standard fungicide Vitavax 200 FS (B.A.S. karboxin and thiuram) was used. After harvesting 100 tubers from each plots was collected and put in storage. The analysis of tuber infestation by Rhizoctonia solani and Helminthosporium solani was made after harvesting (September) and later every 3 months during storage period (December, March). The percent of diseased tubers in tested sample and also infestation degree of bulbs using 5-degree scale was estimated. The received results of investigations ascertained, that all tested preparations during potato vegetation influenced on lower (in comparison with control) degree of bulbs infestation by sclerots of Rhizoctonia solani and the mean degree of infestation by Helminthosporium solani. Moreover the percent of diseased tubers infected by pathogens with tested preparations combination was significant lower than in control.     

A Simple and Fast Extraction Procedure to Obtain Amplifyable DNA from Ralstonia (Pseudomonas) Solanacearum and Clavibacter michiganensis ssp. sepedonicus Inoculated Potato Tuber Extracts and Naturally Infected Tubers to Conduct a Polymerase Chain Reaction (PCR)

A modified NaOH alkaline boiling procedure using a mixture of lysozyme and proteases combined with minimized TRIS/HCl/BSA buffer volume was applied to extract amplifyable DNA from the two quarantine bacteria Ralstonia (Pseudomonas) solanacearum and Clavibacter michiganensis ssp. sepedonicus artificially added to potato tuber extracts of a low and a high starch potato variety. A PCR detection threshold of 10⁴−10⁵ colony forming units per ml extract of each quarantine bacterium was obtained by using the two potato varieties, the high starch potato variety resulting in a lower detection threshold.
Similar sensitivities could be obtained from potato tubers naturally infected with both quarantine bacteria. When comparing a published DNA extraction procedure suitable for Clavibacter michiganensis ssp. sepedonicus with the alkaline extraction the latter is much faster and simpler with similar detection thresholds and representing an inexpensive method to obtain suitable template DNA for routine PCR tests.